Project Summary/Abstract Over the last decade, much effort has been made to define the relationship between stroma physical properties and cancer malignancy. Notably, stiffer tissue is known to constitute a highly favorable environment for tumor emergence and growth. Elevated extracellular matrix (ECM) stiffness affects several processes associated with tumor progression, including the cell response to growth factors and cell migration. Conversely, tumors also exhibit a marked change in the alternative splicing profiles of several key proteins involved in the cell response to growth factors and cell migration. We recently reported that the mechanical properties of the ECM also influence splicing events, revealing a previously unknown regulatory mechanism that could potentially influence tumor progression. This mechanism was proven to be dependent on cell contractility. Interestingly, elevated cell contractility is also a hallmark of aggressive tumor cells (ref) and we and others have shown that cell contractility is also required for proper growth factor receptor activation (ref). Furthermore, my previous doctoral work and my preliminary data demonstrate that specific proteins, such as keratin intermediate filaments and tissue transglutaminase 2, which are differentially expressed in tumor cells, can act as mechanoregulators and as such, can modulate the contractility of cells. By understanding the specific mechanisms that control the altered state of tumor cell contractility and its influence on alternative splicing regulation, this work will reveal an entirely novel strategy in designing cancer therapeutics. I propose to merge prior training in physics and cellular and molecular biology with new training in biomedical engineering and murine tumor models to further investigate and uncover the role of tumor mechanics in modulating key alternative splicing mechanisms to drive tumor progression. In Aim 1 (K99 phase), I will evaluate the effects of altered tumor cell mechanoregulation on the regulation of alternative splicing and the progression of a metastatic phenotype. I will focus on keratin and tissue transglutaminase 2 which are known to be differentially expressed in tumor cells compared to their normal counterparts. In Aim 2 (K99/R00 phase), I will elucidate how alternative splicing of focal adhesion proteins can influence a cell?s ability to adapt to mechanical cues from the extracellular matrix by using a combination of targeted siRNA molecular tools and an engineered matrix of tunable stiffness. These will be further investigated using in vivo mouse tumor models and genomic tools. In Aim 3 (R00 phase), I will investigate the functional crosstalk between growth factor signaling, matrix stiffness, and alternative splicing regulation in the context of tumor progression, combining the in vitro and in vivo training I will have acquired during the mentoring phase of this award, to point toward a targeted therapeutic approach specific to tumor cells within their stiff microenvironment. This Transition to Independence proposal describes research and career development activities, including mentoring, networking opportunities, conference attendance and course training, which will establish me as a competitive candidate for an independent faculty position and will aid in my development of an innovative, successful research program in the field of mechanobiology at the intersection of Physical Sciences and Oncology. These activities will be mentored by Drs. Cynthia Reinhart-King (primary mentor) and Richard Cerione (co-mentor) at Cornell University, a world-class research institution and leader in field of Physical Sciences and Oncology.